Several problems are presented that are directly related to the mechanism for enzymatic catalysis of aldose-ketose isomerization reactions, glycosyl transfer reactions and aldol condensation reactions. The solutions to these problems have eluded investigations employing some of the most sophisticated experimental methods of mechanistic enzymology. This proposal describes how these problems may be resolved by an application of the tools and principles of physical organic chemistry. Three different projects are described. (1) It is not known whether the nonenzymatic or enzyme-catalyzed isomerization reactions of sugars proceed by a hydride or a proton transfer mechanism, or if general acids and metal ions are effective catalysts of these reactions. Questions about the nonenzymatic reactions will be resolved by a study of the isomerization of glyceraldehyde and a related substrate. Proton and hydride transfer reactions win be distinguished by use of deuterium or tritium-labelled compounds. Catalysis by general acids and metal ions will also be studied. (2) It is not known whether the glycosyl transfer reactions catalyzed by beta-galactosidase proceed by a mechanism with discrete chemical steps for every change in bonding, or by a concerted mechanism where two or more changes in bonding occur in a single step. These distinctions are necessary for the definition of the roles of the catalytically essential amino acid residues Glu-461 and Tyr-503. Stepwise and concerted mechanisms for the addition of nucleophiles to the galactosyl-enzyme intermediate (E-Gal) will be distinguished in a study of the reactions of nucleophilic anions. Stepwise and concerted mechanisms for general base-catalyzed cleavage of the acylal linkage will be distinguished by a determination of the effect of changing the basicity of the nucleophile on the reactivity of E-Gal toward alkyl alcohols and phenols, and the solvent deuterium isotope effects on these reactions. The transition state for the general base-catalyzed reaction will be characterized on a two-dimensional reaction coordinate profile. (3) The barrier for the addition reaction of an enolate ion to a carbonyl group in water will be determined, in order to assess whether it is advantageous for enzyme catalysts (e.g., aldolases) to stabilize the transition state for this reaction. The effectiveness of buffer acids as catalysts of addition of enolates to the carbonyl group will also be determined. It has been demonstrated on numerous occasions that studies on enzyme mechanisms may provide information critical for drug design (enzyme inhibitors), to the understanding metabolic diseases, and to the resolution of other health-related problems.